The mammalian intestinal mucosa is in a constant state of renewal, characterized by active proliferation of stem cells localized in the crypts, progression of these cells up the crypt-villus axis with cessation of proliferation and subsequent differentiation and apoptosis. A delicate and precise balance of proliferation differentiation -- apoptosis must exist in the gut mucosa to maintain normal intestinal homeostasis. The major focus of our collaborative group has been the analysis of mechanisms regulating intestinal proliferation, differentiation and apoptosis. With support of this grant, we have made great strides in our better understanding of the molecular mechanisms contributing to intestinal cell differentiation and apoptosis. We have shown that the phosphatidylinositol-3 kinase (PI3K) pathway plays an important role in intestinal differentiation and proliferation;inhibition of PI3K by chemical inhibitors or overexpression of PTEN, a tumor suppressor, enhanced enterocyte differentiation. Furthermore, PI3K inhibition leads to upregulation of the apoptosis-related protein TRAIL which is localized to the differentiated cells of the colon and small bowel;overexpression of PTEN in prostate cancer cells induces TRAIL expression through activation of the FOXO family of forkhead transcription factors. Recently, our studies have identified a novel interaction between PI3K/Akt and NF-_B via transcriptional regulation of PTEN. Based on our findings, the central hypothesis of this proposal is that intestinal cell differentiation is regulated by signaling mechanisms involving the PI3K/Akt/PTEN and NF-kB pathways acting on downstream effectors (eg, FOXO and TRAIL proteins);novel interactions between these pathways exist that further add to the complex, but highly regimented, regulation of intestinal homeostasis. To examine this hypothesis, we have planned experiments with the following Specific Aims: 1) to further delineate the role of the PI3K/Akt/GSK-3 pathway in gut differentiation, 2) to define novel mechanisms of PTEN regulation in the intestine, and 3) to examine downstream targets of the PI3K/PTEN pathway in the intestine. The studies in this current proposal are direct extensions of our previous findings and are designed to define, in a systematic fashion, the molecular mechanisms and signaling events regulating the process of normal intestinal differentiation and apoptosis. Delineating the cellular factors and signaling pathways regulating gut differentiation is crucial to our understanding of not only normal gut development and maturation, but also aberrant gut growth.